Frontiers in Laboratory Medicine 1 (2017) 192–199

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Frontiers in Laboratory Medicine

j ou rna l homepage : www.kea ipub l i sh ing . com/FLM; www. f ron t l abmed . com

Graphene quantum dots in biomedical applications: Recent advances andfuture challenges

Fei Chen a,b,1, Weiyin Gao c,1, Xiaopei Qiu b,d, Hong Zhang b,d, Lianhua Liu b, Pu Liao f,⇑, Weiling Fu e,⇑,Yang Luo b,d,⇑aChongqing Medical University, Fifth Clinical College of Chongqing Medical University, Chongqing 400016, ChinabBioengineering College of Chongqing University, Chongqing 400044, ChinacDepartment of Urology, The Second Affiliated Hospital of Nanchang University, Nanchang, 330006, ChinadDepartment of Clinical and Military Laboratory Medicine, School of Medical Laboratory Science, Southwest Hospital, Third Military Medical University, Chongqing 400038, ChinaeDepartment of Laboratory Medical Science, Southwest Hospital, Third Military Medical University, Chongqing 400038, ChinafClinical Laboratory Center of Chongqing, Chongqing 400014, China

a r t i c l e i n f o

Article history:Received 21 December 2017Received in revised form 24 December 2017Accepted 26 December 2017Available online 8 January 2018

Keywords:Graphene quantum dots (GQDs)ImmunosensorsImagingDrug deliveryToxicity

https://doi.org/10.1016/j.flm.2017.12.0062542-3649/� 2018 Chinese Research Hospital AssociaThis is an open access article under the CC BY-NC-ND l

⇑ Corresponding authors at: Bioengineering CollegeE-mail addresses: liaopu@sina.com (P. Liao), fwl@t

1 these authors contribute equally to this work.

a b s t r a c t

Graphene quantum dots (GQDs)-based nanohybrid materials have gained great attention in multipleresearch applications, particularly in biomedical fields due to their unique physicochemical propertiesand outstanding biocompatibility compared to other nanomaterials. In this review, we focus on the mostrecent emerging developments including synthesis methods, in vivo imaging and in vitro biosensingapplications. We also discuss these unresolved problematic and controversial issues facing their biomed-ical applications. Consequently, trends in approaches to improve the analytical performance of GQDs-based nanomaterials have also been put forward.� 2018 Chinese Research Hospital Association. Production and hosting by Elsevier B.V. on behalf of KeAi.This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-

nd/4.0/).

Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193Synthetic considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Preparation and physicochemical properties of GQDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

Biomedical applications of GQDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

GQDs for in vitro biomedical experimental systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

GQD-based immunological assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194GQD-based nucleic acid assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

GQDs for in vivo imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195GQD-based platforms for drug delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

Toxicity consideration of GQD materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196Conclusions and future perspectives (Unresolved problems, challenges and controversies) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

tion. Production and hosting by Elsevier B.V. on behalf of KeAi.icense (http://creativecommons.org/licenses/by-nc-nd/4.0/).

of Chongqing University, Chongqing 400044, China (Y. Luo).mmu.edu.cn (W. Fu), luoy@cqu.edu.cn (Y. Luo).

Fig. 1. Preparation and properties of GQDs (A) Synthesis process under ultraviolet irradiation. The light yellow GQD aqueous solution was obtained. Reproduced withpermission from Ref. 3. Copyright 2017 American Chemical Society. (B) Preparation of GQDs by microwave-assisted hydrothermal method. Reproduced with permission fromRef. 24. Copyright 2012 American Chemical Society. (C) Schematic representation of GQDs prepared by solvothermal method. Reproduced with permission from Ref. 28.Copyright 2016 Elsevier B.V. (D) Synthesis process of the photoluminescent GQDs by using hexa-peri-hexabenzocoronene as materials. Reproduced with permission from Ref.29. Copyright 2011 American Chemical Society.

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Introduction

Graphene quantum dots (GQDs), a member of the graphenefamily, known as a novel type of zero-dimensional luminescentnanomaterials, are small graphene fragments to cause excitationconfinement in 3–20 nm particles and quantum-size effect.1 GQDsexhibit extraordinary optoelectronic properties as well as excellentbiocompatibility and low-cost preparation methods, by which theyhold potentials in replacing those well-knownmetal chalcogenidesbased quantum dots.2 Besides, the p–p bonds below and above theatomic plane give graphene exceptional thermal and electrical con-ductivity, compared with conventional semiconductor quantumdots, making GQDs possess their favorable attributes withoutincurring the burden of intrinsic toxicity(Fig. 1A).3–7Thequantum-confinement effect and the variation in density and nat-ure of sp2 sites available in GQDs make their optical propertiesgreatly depend on their size so that the energy band gap of GQDscan be tuned by modulating their size.8

Over the past few decades, quantum dots with steadily increas-ing research have been occupied a special status in nanomaterialfields and have made considerable progress, but in recent years,graphene based nanohybrid has captured more interest and imag-ination in scientific research areas.9 Owing to its extraordinarymechanical properties, biocompatibility, transparency and electri-cal conductivity, Graphene quantum dots GQDs has obtained arapid growing as breakthrough tools for multipurpose in variousfields of science including photonics, composites, energy, and elec-tronics. Meanwhile, GQDs-based nanomaterials have alreadyshown a promising future in biomedical fields, particularly fordiagnostics,10–14 drug delivery,15 near-infrared (NIR) light-induced photothermal therapy,16–18 in vitro and in vivo bioimag-ing.19–21 In addition to those fundamental applications as

mentioned above, Ding et al. have reported a GQD-based anti-cancer drug carrier and a signaler for indicating drug delivery,release, and response by providing distinct fluorescence signalsat different stages.22 This research of multifunctional GQDs pro-vides a new direction in future biomedical applications.

In this article, the applications of GQDs in biomedical fieldsincluding biomolecule detection, bioimaging, drug delivery andcancer therapy will be reviewed. Future perspectives and chal-lenges for applying GQDs-based materials in nanomedicine fieldswill also be covered.

Synthetic considerations

Preparation and physicochemical properties of GQDs

In order to be explored in biomedical applications, GQDs arecommonly synthesized by a multi-step synthetic and preparatoryprocess ensuring their solubility and biocompatibility. Those syn-thesis methods can be briefly classified into two categories: top-down methods and bottom-up routes. The top-down methods thatdominated in nanoscience by cutting down large graphene sheetscarbon nanotubes (Fig. 1B),23,24 carbon fibers or graphite into smallpieces of graphene sheets are the most suitable for mass produc-tion; on the contrary, the bottom-up routes that require smallmolecules to be starting materials for GQDs buildup are particu-larly appropriate for controlling the size of GQDs but require mul-tistep organic reactions and purification at each step. By thesereasons, those top-down approaches exemplified as nanolithogra-phy technique, acidic oxidation, hydrothermal or solvothermalmicrowave assisted, sonication-assisted, electrochemical,photo-Fenton reaction, selective plasma oxidation, and chemical

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exfoliation methods have been widely used to synthesize GQDs;while for the bottom-up techniques, using oxygen-containing aro-matic compounds as starting materials polymerized under ultravi-olet irradiation method,3 carbonization as starting materialsthrough microwave-assisted hydrothermal method,25 fullerenesas starting materials through ruthenium-catalyzed cage-opening,26 hydrogen peroxide and expanded graphite as startingmaterials in a one-step solvothermal method27 and using unsubsti-tuted hexa-peri-hexabenzocoronene as the carbon source throughthe process of surface functionalization(Fig. 1C),28 oxidization, car-bonization and reduction successively have been utilized to pre-pare GQDs recently(Fig. 1D).29 On the basis of these two classicsynthetic methods, GQDs with particular physicochemical proper-ties can be prepared for different applications. Hence, the next sec-tion will focus on the properties of GQDs briefly.

Owing to these specific routes for synthesis, GQDs with excel-lent electrical conductivity, optical transparency, and mechanicalstability have mostly been utilized for electronic, electrochemical,and optical applications. It has the property of extremely highintrinsic mobility of charge carriers.30 Among various nanogra-phene materials, GQDs have attracted increasing research interestbecause of its nonzero band gap induced by quantum confinementand edge effects.31 Meanwhile, the high solubility of the GQDs incommon solvents is also very important for experimental studies,surface modified GQDs may contain carboxylic acid groups at theiredges that make them highly soluble in water and suitable for fur-ther modification with other functional molecules such as organic/inorganic and biological components.32 Li et al. proposed a three-step mechanism to elucidate the complex aggregation of GQDs inaqueous solution, which enables us to simplify the interpretationof experimental results for applying readily available, versatileensemble characterization techniques and together with the struc-tural uniformity.33

Biomedical applications of GQDs

GQDs for in vitro biomedical experimental systems

GQD-based immunological assayImmunosensors are fast and simple analytical methods for the

determination of many clinical diseases and biochemical composi-tions that relies on classical antibody-antigen (Ab-Ag) interactions,which supply a hopeful strategy for clinical diagnostics, due totheir specific and sensitive properties. Conventionally,immunosensors are based on recognizing the complexity of anantigen with a specific antibody coupling, one of which could beimmobilized on the solid substrate for observation, afterward a sig-nal change will occur upon formation of an antigen–antibody com-plex. Highly-sensitive immunosensors can thus be constructedapplying enzymatic reactions involving fixing enzyme-labeledantigen.34 The beneficial structural and compositional synergy ofgraphene allows GQDs to be excellent materials for fabricating var-ious immunosensing platforms. Immunosensors can be classifiedinto electrochemical immunosensors,35 amperometricimmunosensors,36 piezoelectric immunosensors,37 thermometricimmunosensors or magnetic immunosensors, according to thetype of transduction.

Electrochemical immunosensors. Electrochemical immunosensorshave gained much research concern because of the integration ofadvantages of label-free and antigen–antibody interaction at thesurface of the detection device, by which any change in potentialthat reflects the existence of specific protein or peptide could befinally measured. A functionalized and ultra sensitive electrochem-ical immunosensor based on the nitrogen-doped graphene quan-

tum dots (N-GQDs) supported PtPd bimetallic nanoparticles(PtPd/N-GQDs) was developed by Yang and coworkers for thedetection of carcinoembryonic antigen (CEA), demonstrating awide dynamic range ranging from 5 fg/mL to 50 ng/mL with alow detection limit of 2 fg/mL (S/N = 3) for the detection of CEA(Fig. 2A).3 Meanwhile, GQDs-based immunosensors for detectionof a cardiac biomarker in human heart attack have arousedresearchers’ interest recently. In a GQDs fluorescence resonanceemergency transference (FRET) based biosensor for detection ofcardiac Troponin I (cTnI), results demonstrated higher specificity,the lower limit of detection (0.192 pg/mL) and less time (10 min)comparing to traditional detection method(Fig. 2B).38 Similarresults have been obtained in another study of cTnI detection usingGQD-PAMAM nanohybrid modified gold (Au) screen printed elec-trode (Fig. 2C).39 Electrochemical immunosensors based on GQDsare the most heated research orientation in recent years, and theenthusiasm for research on it will not be easily extinguished.

Amperometric immunosensors. Comparing with other kinds ofimmunosensors as previously mentioned, amperometricimmunosensors are far more broadly investigated due to theireasyfabrication, miniaturization, robustness, and cost-effectiveness.40 Huang et al. developed a simple amperometricimmunosensor based on TiO2-graphene, chitosan and goldnanoparticles (AuNPs) composite film-modified glassy carbon elec-trode (GCE) for protein detection. The negatively charged AuNPscan be adsorbed on the positively charged chitosan/TiO2-graphene composite film by electrostatic adsorption, and thenused to immobilize a-fetoprotein antibody for the assay ofa-fetoprotein(AFP). By using this strategy, a wide detection range(0.1–300 ng mL � 1) with the correlation coefficients of 0.992–0.994 for model target AFP is obtained. The limit of detection is0.03 ng/mL at a signal-to-noise ratio of 3.41 The research on amper-ometric immunosensors has resemblance with that on electro-chemical immunosensors.

Other types of immunosensors. Coupling immunoassay techniqueswith surface plasmon resonance (SPR) technology are the princi-ples of optical immunosensors. A change in refractive index ofthe medium upon interaction of Ag (tumor marker) with specificantibodies immobilized on the sensor surface can be measured.Alternatively, antibodies could be immobilized on the surface ofan optical fiber so that any change in refractive index, fluorescenceor luminescence correlated with a number of antigens interactingwith the antibodies could be measured.42 The principle of piezo-electric immunosensors is commonly utilized by evaluating thechange in frequency of oscillation as result of a change in masson the interaction of antigens with antibodies immobilized onquartz crystal.43 These kinds of immunosensors are not wellapplied in biomedical fields mainly due to their high cost, difficultyin mass production and problems with mechanical and electro-magnetic interference.

GQD-based nucleic acid assayAs they provide a simple, accurate and cheap platform for DNA

detection, electrochemical detection methods based on GQDsbiosensors were also extensively adopted in the various nucleicacid assay. In addition, electrochemical DNA sensors can improvethe immobilization of single-stranded DNA (ssDNA) probesequences on a mass variety of electrode substrates.44,45 Qianet al. introduced a method to achieve the analysis of the low con-centration of DNA by taking advantage of excellent biocompatibil-ity and powerful fluorescence of GQDs, one base pair DNAmismatch specificity and unique FRET between carbon nanotubesand GQDs.46

Fig. 2. Three kinds of electrochemical immunosensors based on GQDs. (A) The label-free electrochemical immunosensor and the preparation procedure of PtPd/N-GQDs@Au.Reproduced with permission from Ref. 3. Copyright 2017 American Chemical Society. (B) The reaction between cardiac antibody and functionalized GQDs with sensingmechanism of FRET (a) Bioconjugation of the monoclonal antibody anti-cTnI with afGQDs using EDC–NHS chemistry. (b) Schematic mechanism of immunosensing based onspecific interaction of anti-cTnI/afGQDs with graphene. Reproduced with permission from Ref. 38. Copyright 2016 Elsevier B.V. (C) Schematic illustration of theimmobilization of the cardiac troponin I antibody (anti-cTnI) probe on the Au/GQD/PAMAM nanohybrid electrode and electrochemical detection of cTnI. SPE: Screen printedelectrode (CE: counter electrode, WE: working electrode, RE: reference electrode). Reproduced with permission from Ref. 39. Copyright 2016 Elsevier B.V.

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Hu et al. constructed a double-stranded DNA structure by thehybridization of thiol-tethered oligodeoxynucleotide probes (cap-ture DNA) that was assembled on the gold electrode surface withtarget DNA and aminated indicator probe (NH2-DNA). After theconstruction of the double-stranded DNA structure, activated car-boxyl groups of GQDs assembled on NH2-DNA could be appliedfor DNA recognition. GQDs were used as a new platform for horse-radish peroxidase immobilization through the noncovalent assem-bly. With the integration of GQDs and enzyme catalysis, theproposed biosensor could detect miRNA-155 from 1fM to 100 pMwith a detection limit of 0.14fM.47 At present, development ofDNA/RNA sensors is receiving considerable attention in biomedicalfield with the scope of improving the sensitivity and selectivity ofsensors.

GQDs for in vivo imaging

On account of relatively high quantum yields with high molarextinction coefficients, broad absorption with narrow emissionspectra and high photostability, the strong quantum confinementand edge effects stir GQDs wide applications in biological imag-ing.1,48,49 Thus, choosing suitable probes plays a significant rolein bioimaging purposes since that the resolution, sensitivity, andversatility of fluorescence microscopy are mainly depended onthe properties of various fluorescent probes. The novel GQDsderived nanomaterials featured by many advantages comparingwith a traditional imaging modality, such as only a small numberof GQDs are needed to generate the signal due to the highly stableand bright fluorescence. Meanwhile, GQDs with the near-infraredreflectance emission property are promising candidates for theimaging of deeper tissue samples.30 Based on the above reasons,applying GQDs as contrast agents for in vivo imaging has been

the area of high expectations and recurring attention. The near-infrared emitting window shows great advantages for biomedicalimaging because of the low tissue absorption and reduced lightscattering in more than 650 nm wavelengths’ region. Near-infrared graphene quantum dots-based nanoprobe for ascorbicacid (AA) detection in living cells was reported by Tan and co-workers. They announced that GQD possessed good two-photonfluorescence properties showing a emitted near-infrared reflec-tance (660 nm) upon excited with 810 nm femtosecond pulsesand a two-photon (TP) excitation action cross-section of25.12GM. They were then employed to construct a TP nanoprobefor detection and bioimaging of endogenous ascorbic acid in livingcells. In this nanosystem, near-infrared reflectance GQDs, whichexhibited lower fluorescence background in the living system toafford improved fluorescence imaging resolution, were acted asfluorescence reporters.50

Doping GQDs with heteroatoms has recently been the trendsbecause of the quickly expanded research needs in cell imaging,it can be an effective way to modulate the band gap, tune elec-tronic density and chemical activity of GQDs, which endows theheteroatoms-doped GQDs new optical phenomena and unexpectedproperties for practical applications. A N-GQD was composed forphotodynamic antimicrobial therapy and bioimaging, the resultshowed the intrinsic luminescence properties of the N-GQD inNIR region and high photostability enable it to be exploited as apromising contrast agent to track bacteria in bioimaging.51

GQD-based platforms for drug delivery

To improve the water solubility and the specific targeting ofdrugs, various nanocarriers have been developed. MultifunctionalGQDs usually serve as drug carriers and targeted cellular imaging

Fig. 3. GQDs based nanocomposites in drug delivery. (A) TEM image of GQDs. (B) FTIR spectra of GQDs and GQD–FA. (A and B) Reproduced with permission from Ref. 53.Copyright 2014 Elsevier B.V. (C) TEM image of N-GQDs. (D) FTIR spectra of (a) N-GQDs and (b) MTX-(N-GQDs). (C and D) Reproduced with permission from Ref. 18. Copyright2017 Elsevier B.V.

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simultaneously, which can be used in cancer therapy. Drug deliv-ery systems could be visualized by using organic fluorophoresand semiconductor quantum dots to understand the cellularuptake, while we can easily monitor movement in the cells in realtime without employing external dyes considering the inherentfluorescence of GQDs.52 An experiment showed that synthesizedfolic acid (FA)-conjugated GQDs could be utilized to load the anti-tumor drug doxorubicin (DOX). The fabricated nanoassembly canbe unambiguously discriminate cancer cells from normal cellsand efficiently deliver the drug to target cells. The inherent stablefluorescence of GQDs enables real-time monitoring of cellularuptake of the DOX–GQD–FA nanoassembly and the consequentrelease of drugs. The nanoassembly is specifically internalizedrapidly by HeLa cells via receptor-mediated endocytosis, whereasDOX release and accumulation are prolonged. In vitro toxicity, datasuggest that the DOX–GQD–FA nanoassembly can target HeLa cellsdifferentially and efficiently while exhibiting significantly reducedcytotoxicity on non-target cells (Fig. 3A and B).53

A new hybrid nanosystem powerful multimodal tool for thetreatment and imaging of cancer has been reported that GQDswere used as a multifunctional nanocarrier to load Gadoliniumtexaphyrin and lutetium texaphyrin for biological redox therapy-enhanced photodynamic and photothermal therapy, which exhi-bits tumor-responsive deep-red fluorescence and enhanced T1-weighted MRI that enable imaging of the tumor during treat-ment.17 GQDs sheets can increase their drug loading capacity viatheir unique structure of two faces and edges, Fatemeh Khodadadeet al. synthesized 10 nm size nitrogen-doped GQDs(N-GQDs) with10 graphitic layers loading methotrexate(MTX) to construct a drugdelivery system, the results revealed that GQDs as nanocarriershad stronger anti-tumor cells activities since it can prolong thecytotoxic effects of loaded drug(Fig. 3C and D).18 The receptor-mediated endocytosis of GQDs promised a more accurate andselective cancer diagnostic approach.

Toxicity consideration of GQD materials

The toxicity of nanomaterials is one of the major challenges fac-ing their applications in biotechnology. Studies on the cytotoxicityof graphene-based materials have stated briefly that GQDs with aless than 50-nm side edge caused no obvious toxicity to a seriesof cells (Fig. 4A).54 Since single-dosing experiment had no obviousaccumulation and mostly presented low toxicity of nanomaterials,multiple-dosing which simulated clinical drug administration wasapplied to the study of in vivo toxicity in order to further investi-gate the biosafety of GQDs.55 Nurunnabi et al. performed in vitrocytotoxicity studies on carboxylated GQDs and observed no toxic-ity (Fig. 4B).56 Peng et al. have found that nano-sized graphene oxi-des did not lead to serious acute cytotoxicity to HeLa cells at aconcentration of 40 lg/mL.57 Li et al. observed no distinct celldeath by incubating graphene oxide nanoparticles with gastriccancer cells and skin cells at a dose up to 100 lg/mL (Fig. 4C).58

With the cytotoxicity studies of GQDs, it is important to assessthe potential compromises of GQDs to DNA damage since thereis a close correlation between DNA damage and variation or cancer.In a research reported by Wang et al., genotoxicity of GQDs to NIH-3 T3 cells was inquired by analysis of flow cytometry for DNA dam-age related protein activation while the GQD induced ROS genera-tion was studied as a potential cause for DNA damage. The cellularuptake of GQDs, as well as cell death and proliferation of NIH-3 T3cells treated with GQDs, was also studied to assess the cytotoxicityof GQDs.59 Yuan et al. investigated the cell distribution of threeGQDs modified with different functional groups (NH2, COOH, andCO-N (CH3)2, respectively) and compared their cytotoxicity inA549 and C6 cells, no visible mortality and apoptosis or necrosisincreases resulted from the treatment of the three GQDs even atthe concentration of 200 lg/mL, the results manifested that whenmodified with different chemical groups, GQDs still possessedexcellent biocompatibility and low cytotoxicity to cells, which

Fig. 4. Researches on toxicity of GQDs. (A) Results all suggest no obvious toxicity of GQD-PEG to HeLa cells at GQD concentration as high as 160 mg/mL. Reproduced withpermission from Ref. 54. Copyright 2014 Elsevier Ltd. (B) Cellular cytotoxicity of Fluo–G, PEG–G, and GO to HeLa cells(incubated with different concentrations of graphenederivatives for an additional 24 h in fresh medium). Reproduced with permission from Ref. 56. Copyright 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (C)a. Cellviability of NIH-3 T3 cells studied by MTT method and the cells were treated in cell culture media with GQDs (0, 5 and 50 lg/mL) for 3 and 24 h, respectively. b. Averagenumbers of NIH-3 T3 cells after the addition of GQDs for 0, 24, and 48 h post incubation. (C) Reproduced with permission from Ref. 58. Copyright 2013 The Royal Society ofChemistry.

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may make them more promising in bioimaging and other biomed-ical applications.60 Markovic et al. analyzed GQD-mediated photo-dynamic cytotoxicity in terms of molecular mechanisms, showingthat the induction of oxidative stress generated in vitro photody-namic cytotoxicity of GQD, and subsequent activation of bothapoptosis and autophagy programmed cell death.61 But humanbreast cancer studies have indicated that they are non-toxic mate-rials since GQDs can rapidly get into the cytoplasm and do notinterfere with cell proliferation.62 These results from the cytotoxi-city studies at the cellular level are in favor of GQDs for biomedicalapplications. But as a precaution, more attention should be paid tothe safety of GQDs by studying their intracellular and in vivo meta-bolic pathways of toxicity, cellular uptake mechanisms.

Conclusions and future perspectives (Unresolved problems,challenges and controversies)

One of the key difficulties facing researchers aiming to developGQDs for nanomedical applications is to obtain high-quality prod-ucts, the existing synthesis method generally allows small-scaleproduction of GQDs which have a wide size distribution. It is nec-essary to find easy purification methods and seek out novel meth-ods to achieve a high yield that does not require the removal ofstarting materials. Since the size and shape have a massive impacton the physicochemical properties of GQDs, mass theoretical andpractical research must focus on improving synthetic method ifthe GQDs based fluorescence detection methods gradually replacethe traditional detection technique used in laboratory inspection.63

More studies concerning the application of GQDs basedimmunosensing are needed compared with the non-

immunosensing. Because of the small number of research onimmunosensors based on GQDs, more techniques should beinvolved in this area and novel protocols for detection of cell lines,cancer biomarkers, and disease should be developed, since that thesynthetic of the new technologies will bring significant input toultra sensitive immunosensors relevant to diagnostics, and therapyof cancer.

Understanding the photoluminescence (PL) properties of GQDsis still poor, although some possible mechanisms have been pro-posed, such as size effect, surface modification, and doping withother elements. In spite of the achievement of GQDs with differentcolored PL properties, including PL in the near-infrared region, thequantum yields of most GQDs are still at a low level, so theimprovement of GQDs is imperative because of their restrictedapplication in immunosensing from their lower quantum yields.Metal-enhanced fluorescence may be considered for quantum-yield escalation and a study has proved its possibility. Althoughadvances are exciting and encouraging, the use of GQDs forimmunosensing applications is still in infancy, with a lot of chal-lenges remaining. A new strategy for surface modification isneeded to be developed for application in immunosensing. Withtheir uniform size, excellent PL and high quantum yields, GQDswill no doubt be used in more creative applications. In summary,GQDs emerge as a novel nanomaterial platform for immunosens-ing, effective collaboration between multiple disciplines includingchemistry, physics, biology, and medicine must be implemented.

In this review, we have summarized recent research progress ofgraphene quantum dots based nanomaterials, focusing on theirsynthesis, typical properties, and biomedical applications includingin vitro and in vivo, we also share a brief introduction about the

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in vivo toxicity of graphene quantum dots mainly based on celllevel. Finally, we conclude that there is a promising future for fur-ther advances and developments of GQDs based nanomaterials.

Acknowledgement

This work is partly supported by Grants from the National Nat-ural Science Foundation of China (81572079, 81371899 and81601854), the Science Fund for Distinguished Young Scholars ofChongqing (CSTC2014JCYJJQ10007), the China Academy of Engi-neering Physics (WSS-2014-09), and the Southwest Hospital(SWH2016ZDCX1017, SWH2016JQFY-01).

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